Collagenase (clostridiopeptidase A, EC 3.4.24.3) is a proteolytic enzyme that hydrolyzes collagen proteins, both in their native and denatured (gelatins) form. No other enzyme is capable of cleaving native collagen, due to its distinct amino acid composition (presence of a numerous amount of imino acids, represented by tripeptide Gly-Pro-X, where X is, frequently, proline or hydroxyproline), and macrostructure (complex triple-helix structure).
Collagenase, isolated or as the main component of compositions comprising other proteolytic enzymes, has been widely used since the 70's in the treatment of several diseases and pathological conditions. All diseases and conditions in which collagenase is administered are associated with the excessive deposit of collagen and the erratic accumulation of fibrous tissue rich in collagen. Such diseases and conditions are denominated collagen-mediated diseases, and the local application of compositions containing collagenase is one of the possible alternative treatments to surgical intervention. The use of collagenase compositions for the treatment of necrotic tissue debridement (burns, skin lesions, etc.) has also been approved by regulatory agencies, showing surprising results on improving scarification processes.
Some therapeutic applications of collagenase compositions are: burn treatment, with a concomitant beneficial effect over tissue regeneration; enzymatic debridement of wounds and treatment of infected wounds and ulcers (skin and decubitus ulcers); herniated intervertebral disc disease treatment; selective lysis of collagen fibers of the eye's vitreous body; Dupuytren's disease treatment; Peyronie's disease treatment; adhesive capsulitis treatment; lateral epicondylitis treatment, Carpal Tunnel Syndrome and plantar fasciitis; regeneration of damaged nerves; also being widely used in medical specialties such as plastic surgery and in several dermatological and aesthetic procedures, for example, in the treatment of cellulites and scars, such as acne, keloid and other hypertrophic scars through intralesional injection of purified collagenase.
Other studies have shown the use of collagenase compositions in rheumatoid arthritis, metastasis, angiogenesis and cirrhosis.
Collagenases have also been described as important tools in specific organ transplantation i.e. isolation of pancreatic islets of Langerhans, microvascular endothelial cells, hepatocytes and chondrocytes (WO9824889). Tissue dissociation mediated by collagenolytic enzymes is a critical step in several cell isolation procedures.
The collagenases used in the majority of the therapeutic applications described above are obtained from supernatant, purified or not, of bacterial cultures, most specifically of Clostridium histolyticum cultures. Clostridium histolyticum is an anaerobic, gram-positive and spore-forming cylindrical bacterium. The collagenolytic activity of the extracellular enzymes of such bacteria has been known for over 50 years, when it was isolated for the first time an extracellular enzyme capable of digesting cattle Achilles tendons.
Currently, it is of common knowledge that several microorganism species when cultured in specific conditions are capable of producing collagenase. However, Clostridium sp, most specifically Clostridium histolyticum, is still the main source of collagenolytic and gelatinolytic enzymes for therapeutic application due to its higher enzymatic productivity and activity against several collagen forms and its peptides.
Other collagenase sources for therapeutic use include mammal cells, crustaceans (crabs, shrimp), fungi and other bacteria (Streptomyces, Pseudomonas and Vibrio).
The ability of Clostridium collagenases to digest several kinds of collagen (type I, II, III, VII and X) and gelatin is the major factor that differentiates these proteases from other collagenase sources. Aforesaid clostridial enzymes not only present higher collagenolytic activity when compared to vertebrate collagenases but it also presents an optimum pH for its activity within human physiological pH range.
Studies from late 1950s to mid-1980s showed the existence of several types of C. histolyticum collagenases and characterized their specificity and stability gradually. Bond and Van Wart (1984; Biochemistry, 23: 3077-3085) isolated six different enzymes from commercial collagenase preparations with molecular weights varying from 68 kDa to 130 kDa and isoelectric points between pH 5.5 and 6.5; further, classifying them in two classes according to substrates specificity. Class I—higher activity against high molecular weight collagens (native collagen: intact); Class II—preference for low molecular weight collagen fragments (denatured collagen: gelatins). These activities seem to be complementary, and there is evidence of its synergistic action on native collagen.
In addition to the several classes I and II collagenase isoforms, conventional preparations of collagenase, based on the supernatant of Clostridium histolyticum cultures, contain over 30 different enzymes. The primary constituent of the culture supernatant are the collagenases (classes I and II), however, other important proteases are also present, such as: neutral proteases i.e.: gelatinases, clostripains (clostridiopeptidase B, EC 3.4.22.8), trypsins, elastases and aminopeptidases. Non-proteolytic enzymes including: galactosidase, acetylglucosaminidase, fucosidase, phospholipase, neuraminidase (or sialidase) and hyaluronidase can also be found. Of the aforementioned proteolytic and non-proteolytic enzymes, sialidase, hyaluronidase and collagenase are deeply involved in the degradation process of extracellular matrix. The presence of this “enzymatic combination” is required to obtain compositions suitable to be used as enzymatic debridement agent for skin lesion and organ transplant (i.e. isolation of pancreatic islets).
Furthermore, either purified or recombinant, collagenase alone is inefficient in tissue dissociation due to incomplete hydrolysis of collagen polypeptides and to its limited activity when high concentrations of non-collagen proteins and other macromolecules are found in the extracellular matrix. For such reason, the most used collagenase composition for tissue dissociation and debridement is a crude or partially purified preparation (obtained by few purification steps), since the presence of enzymes which act on native collagen and reticular fibers in addition to enzymes responsible for the hydrolysis of other proteins, polysaccharides and lipids in the extracellular matrix of connective and epithelial tissue are required. The complex composition of crude collagenase preparations brings consequences to the production process, due to difficulties to define expression patterns of the proteolytic enzymes of the final composition.
On the other hand, the use of collagenase compositions for the treatment of collagen-mediated diseases require high purity of the preparations, due to the need of a specific action on digestion of collagen; such treatments are administered through local injections. In these cases, the purification processes of collagenase from the supernatant of C. histolyticum cultures are complex, including numerous steps with different strategies of purification.
In general, the purity of collagenase composition is directly related to the therapeutic application intent. However, regardless of the therapeutic application, a defined process for culturing Clostridium histolyticum is essential to direct the production in order to obtain the proteases of interest in the supernatant, purified or not.
The enzymatic activity and the concentration of proteases in C. histolyticum cultures supernatant vary according to the bacterial strain, culture medium, culture conditions, culture age, cellular density, among other variables. The intrinsic variability of this type of biotechnological process implies in exhaustive quality control analysis of the batches, increasing production costs. Moreover, the loss of efficacy of traditional collagenase over storage, regardless of the storage conditions, is strongly related to the enzymatic composition of the culture supernatant. For such reason, defined culture conditions which direct the production of the desired collagenolytic and gelatinolytic enzymes, in special collagenases, are required.
It is well known that the production of extracellular proteases by microorganisms is strongly influenced by components of the culture medium, especially by carbon and nitrogen sources, and physical factors such as, for instance, pH, temperature, inoculum volume, rate of orbital agitation and incubation time. There is no pre-defined culture media for specific proteases production; each microorganism has its own physicochemical and nutritional idiosyncratic requirements for growth and enzymatic secretion.
Proteins and peptides are the main source of carbon and nitrogen in Clostridium culture, being required for protein expression as well as growth. Therefore, brain-heart infusion (BHI), tryptose, peptone proteose, meat extract, casein and gelatin are commonly found in C. histolyticum culture medium. Not only, inorganic salts, vitamins, glucose, yeast extract, sodium thioglycolate and ferrous sulphate are also found in different concentrations in such culture media.
Since 1930's, several authors stated that animal derived peptones are essential for C. histolyticum growth and enzymatic production.
Later, in the 1950s, MacLennan et al. (1953; J. Clin. Invest., 32: 1317) evaluated how several carbon and nitrogen sources (phytones, peptones, collagen and it's derivatives, and glucose), as well as vitamin and inorganic salts concentration, pH and Fe++ influence C. histolyticum growth and collagenolytic and gelatinolytic enzyme expression (several strains). Ferrous salt and animal derived peptone were defined as crucial for maximum enzymatic yield. The results presented in this study have been considered to determine the “conventional culture medium” for C. histolyticum collagenase production for several decades.
Commonly described as examples of animal derived peptones are: bacto-peptone, peptone proteose, hydrolyzed casein, casein tryptic hydrolysate, among others. Combinations of these components with vegetable peptones have also been described. For example, Bergman et al. (1961; Journal of Bacteriology, 82: 5829) obtained good results culturing C. histolyticum in a culture medium which contains peptone proteose, casein hydrolysate, soy tryptic hydrolysates and vitamins. The authors also demonstrated that, in the absence of inorganic salts, the collagenase productivity is equal to or greater than the one presented by MacLennan et al. (1953), however, with a decrease in the production of azocaseins, which is an interesting fact, since it facilitates later purification procedures (called PTV culture medium).
Besides the importance of animal derived peptones for the growth and enzymatic production in cultures of C. histolyticum, the influence of other components in the culture medium has also been investigated in order to define metabolic patterns for this species, thus improving culturing conditions.
Mead (1971; J. Gen. Microbiol., 67: 47) describes the pattern of amino acids intake in several Clostridium species during its growth, classifying them into groups according to their nutritional needs. In the aforementioned study, Clostridium histolyticum is considered capable of fermenting amino acids, and is sub-classified in the so called Group II (C. botulinum; C. histolyticum; C. cochlearium; C. subterminale). Even though serine, glutamine, glycine and valine are the main amino acids used by these species, their addition in culture medium produces no or little effect on the bacterial growth. However, substitution of partial casein hydrolysate by casamino acids significantly increases the observed bacterial growth rate (culture medium used: vitamins, inorganic salts, tryptophan, cysteine hydrochloride, casamino acids—2 g/L or casein hydrolysate—3% w/v and meat extract). Unfortunately, the author did not analyze the protease production.
More frequent than the amino acids intake, glucose intake as energy source for several microorganisms has been well documented. Particularly, for C. histolyticum the addition of glucose (and other sugars) has been correlated with a decrease in bacterial growth rate (Mead, 1971). Thus despite being initially classified as saccharolytic species, due to their ability to metabolize carbohydrates as energy source, several strains of C. histolyticum are inefficient fermenters of simple sugars such as glucose, maltose, among others. Moreover, a decrease in growth rate is even more prominent when culturing C. histolyticum under aerobic conditions in media containing glucose, suggesting the existence of two inhibiting factors: sugars and oxygen/air.
Not only does glucose negatively influence bacterial growth, it has also been reported that glucose containing media decrease protease expression in Clostridium cultures (C. difficile, for example). This observation suggests that there is a relationship between repression of toxin expression and the composition of culture medium with rapidly consumable carbon sources.
As discussed previously, Clostridium bacterial growth rate and protein expression are affected by nutritional requirements as well as the maintenance of an anaerobic environment. C. histolyticum tolerates few amounts of oxygen, as observed to other anaerobes and also other species of the genus, being considered an aerotolerant microorganism. Despite this, minimal conditions of anaerobiosis must be assured for growth and production of collagenolytic and gelatinolytic protease by C. histolyticum liquid cultures.
Additionally, reducing agents are also considered critical in anaerobic liquid culture. Among the most frequently used agents are: meat digest, glucose, sodium thioglycolate, cysteine, iron salts, metallic iron or a mixture of both.
To this date, it remains unknown which culture medium ingredients influence C. histolyticum growth and protease secretion. There is a consensus only about the importance of animal source ingredients, particularly peptones, to Clostridium histolyticum growth and protease expression, i.e. collagenases.
The conventional C. histolyticum culturing processes present several disadvantages, such as: low yield, low reproducibility and incomplete separation of impurities. Furthermore, one of the major drawbacks of such culture processes is the predominant use of animal derived components to obtain collagenolytic and gelatinolytic proteases for therapeutic use in humans.
The presence of animal derived components in such culture processes offers additional and undesirable potential risk of infections and anaphylactic reactions. A well-known example of disease caused due to interspecific horizontal transmission of pathogens is the bovine spongiform encephalopathy (prion disease; “Mad Cow disease”). Its transmission from the original host (bovine) to man was first recorded in 1993, due to contact with infected physiological fluids and consumption of infected animal meat.
In the last decade, regulatory agencies have been encouraging the pharmaceutical industry to use bacterial culture media comprising only non-animal derived components. However, care must be taken when considering non-animal derived components since these are often processed using animal derived enzymes. Even being a very small fraction of the final culture media, the presence of animal derived enzymes prevents media classification as animal product-free.
The following patents and papers state different animal product-free culture media for Clostridium: WO9854296 (1998, Chiron), WO0105997 (2000, Massachusetts Institute of Technology), WO2005035749 (2004, Allergan), Busta & Schroder (1971, Effect of soy proteins on the growth of Clostridium perfringens. Appl. Microbiol, 22(2), 177-183; Demain et al. (2007, Tetanus toxin production in soy-based medium: nutritional studies and scale-up into small fermentors. Lett. Appl. Microbiol., 45(6), 635-638); Fang et al. (2009, Production of Clostridium difficile toxin in a medium totally free of both animal and dairy proteins or digests. PNAS, vol. 106: 13225-13229).
Usually, soy based and yeast extracts components are able to sustain growth rate as well as protease expression and secretion when used to replace animal derived peptones in the culture media of some Clostridium species such as, C. sporogenes, C. tetanus, C. botulinicum. 
However, particularly to C. histolyticum, there is no animal product-free culture media that stimulate the production of collagenolytic and gelatinolytic proteases in a way at least equivalent to the conventional animal derived media.
The culture media described so far to C. histolyticum present disadvantages such as: presence of animal derived components, high cost associated with its complex composition, low collagenase expression and long culturing periods required to obtain a supernatant with collagenolytic and gelatinolytic activity appropriate for industrial production with therapeutic purposes.
Attempts for obtaining an animal product-free culture medium that would be appropriate for C. histolyticum growth and collagenase production are reported below.
The document WO2007089851 (2007; Auxilium) describes C. histolyticum culture conditions in order to obtain collagenases. Although it describes the production of collagenases by C. histolyticum in an animal product-free culture medium, it still emphasizes that in order to produce collagenases with higher reproducibility, a culture medium comprising animal derived peptone is preferred. Only the animal derived culture medium was capable of producing the collagenases in adequate proportion to maximize its synergistic activity, resulting in a therapeutic benefit. Other than peptones (vegetable and/or animal), the culture medium described above may contain amino acids (glutamine, tryptophan and asparagine), yeast extract, inorganic salts, glucose and vitamins.
The document US20100086971 (2009; Roche) describes a culture medium for C. histolyticum which includes vegetable derived peptone. However, in the US2010008671 document, an animal derived additive—fish gelatin—is considered crucial to the higher collagenolytic activity observed in these culture supernatants. It is emphasized that only the liquid composition containing vegetable peptone with fish gelatin can support greater bacterial growth and collagenolytic activity when compared to the standard culture medium comprising animal derived peptone.
Thus, until the present moment, no culture medium completely free from animal derived components has been able to support an adequate industrial production of C. histolyticum collagenases as demonstrated to conventional culture media containing animal peptones.
For such reason, there is a clear need for developing animal product-free culture media as well as processes for production of C. histolyticum collagenolytic and gelatinolytic proteases.
Therefore, the present invention provides an animal product-free culture medium for bacteria of the genus Clostridium, preferably C. histolyticum, and an industrially adequate process for producing a supernatant comprising one or more collagenolytic and gelatinolytic proteases, in particular collagenases, for therapeutic purposes.